Summary: Signals mediated by the T cell receptor (TCR) and associated signaling molecules regulate both T cell development and the balance between activation and tolerance in both early T cells (thymocytes) and mature T cells. Understanding how TCR signals regulate these processes is essential for understanding autoimmune diseases and designing effective vaccines to pathogens. The antigen recognition ability of T cells is supplied by clonotypic chains (TCRa,b), but the signaling potential is conferred by the CD3 subunits (CD3e, CD3g,and CD3d) and members of the zeta (z)-chain family. The ability of z-chain and CD3 components to transduce signals derives from a consensus sequence (ITAM) present within their intracytoplasmic domains. Each CD3 chain contains a single ITAM, whereas full length z-chain contains three ITAMs. To understand better the role of specific and multiple signaling motifs in immune processes, we have studied genetically altered mice lacking TCR-z chain and/or CD3e. Reconstitution of these "knock out" mice with transgenes encoding genetically engineered variants of z-chain and/or CD3e have led to an in-depth analysis of the role of TCR ITAM multiplicity in T cell development and function. Recently, several key points have been demonstrated. 1)A critical relationship exists between the affinity of the TCR for selecting ligand and the signaling potential of the TCR for both positive and negative selection of the T cell repertoire. 2) Using a murine model of multiple sclerosis, EAE(experimental autoimmune encephalomyelitis, we found that mice with impaired negative selection (reduced numbers of ITAMs) were more susceptible to induction of autoimmune disease than mice whose TCR contain the full complement of ITAMs. 3) Responses mediated by mature T cells are altered if the signaling potential is genetically altered, resulting in greater dependence on costimulatory/adhesion molecules and the type of cytokines generated. 4)The ITAM present in the CD3e chain does not have a specific function in development but rather contributes to TCR function through signal amplification. We are currently constructing mice that lack both CD3e and z-chain and will reconstitute these animals with CD3e and z-chain variant molecules to further investigate the multiple signaling structure of the TCR. We have also investigated how molecules that regulate signaling through the TCR impact on T cell development. We have found that CD5, a molecule that negatively controls signals through the TCR, impacts on T cell development in a manner dependent on the avidity of the TCR for self ligands and the signal strength of the TCR. In a recent collaboration, we are also examining how a TCR adapter molecule, LAT, can regulate T cell development and function in vivo using mutant LAT "knock in" mice. These studies indicate that specific tyronsine residues in LAT differentially control signaling pathways in T cells and modulate functional outcome of TCR signals.